The Notch-1 receptor represent a strong candidate marker of neural stem cells, capable of differentiation along neuronal, astroglial and oligodendrocyte lineages. This research proposal aims to produce a specific, high affinity rat monoclonal antibody (mAb) against the NH2-terminal extracellular domain of murine Notch-1 (Notch.1[ext]) to be used to select Notch-1+ cells from embryonic murine forebrain and early passage cultures by fluorescence-activated cell sorting (FACS). Two strategies will be used to hyperimmunize rats: direct DNA immunization, and repetitive immunization with DNA and recombinant Notch-1[ext] protein. Notch-1 + and Notch-1- cells will be sorted, one cell/well, and evaluated for stem cell properties using a neural stem cell assay. Finally, we will attempt to expand the stem cells from Notch-1 sorted and unsorted pools of primary neurospheres by treatments with recombinant Notch ligand (Delta-1[ext]) and synthetic peptide agonists. Thus, we will establish the feasibility of an approach using monoclonal antibodies against the Notch-1 receptor to select multipotent neural stem cells by FACS, and expand the neural stem cell population, in vitro. In Phase II we intend to produce and commercialize rat anti-Notch-1 mAb, begin molecular characterization studies of Notch-1+ neural stem cells, and expand and bank Notch-1+ stem cells for transplantation studies in mice. PROPOSED COMMERCIAL APPLICATIONS: This phase I application will develop and use monoclonal antibodies against the Notch-1 receptor to select neural stem cells by FACS, and attempt to expand the neural stem cell population, in vitro, with soluble Notch ligand (Delta-1[ext]) and synthetic peptides. Products derived including monoclonal antibodies, Notch-1+ stem cells, synthetic and recombinant Notch-1 agonists will have significant commercial value and societal impact on the fields of stem cell biology, pharmaceutical testing, and transplantation.